Abstract Post-translational modifications on histone proteins play an essential role in regulating chromatin transcription in human cancers. Histone acetylation is associated with active gene transcription and plays a crucial role in tumorigenesis. Indeed, small molecules targeting proteins involved in regulation of histone modifications are being explored as a very promising anti-cancer agents, with a number of compounds currently in clinical trials (e.g. BRD4 inhibitors). Recently, the YEATS domains have been discovered as novel acetyl-histone reader domains. GAS41 was originally found to be amplified in 23% glioblastomas and 80% astrocytoma. Emerging studies strongly implicate GAS41 as an oncogene in Non-Small Cell Lung Cancer (NSCLC). GAS41 is frequently amplified in NSCLC and knockdown of GAS41 or disruption of the interaction with acetylated histones suppresses lung cancer cell growth. We have recently found that GAS41 is a reader of di-acetylated H3 histone. Full-length GAS41 is dimeric in cells and binds di-acetylated H3 with high affinity. Based on this finding we developed suites of biochemical assays suitable for characterization of GAS41 protein-protein interactions and identification of small molecule inhibitors. In this proposal we plan to develop small molecule inhibitors of GAS41 using high throughput screening (HTS) in CCG at the University of Michigan. Small molecule inhibitors of GAS41 will be validated and characterized in a series of biochemical and biophysical experiments. Activity of the most potent compounds will be characterized in cell-based assays to assess the disruption of GAS41 interactions with chromatin and understand mechanism of action. Selected GAS41 inhibitors will be also profiled in a panel of lung cancer cell lines. In summary, we expect to identify highly valuable chemical probe compounds targeting GAS41 protein-protein interactions suitable for mechanistic studies and pave the way towards development of potent in vivo active GAS41 inhibitors.